1. Field of the Invention
The present invention relates to methods of in vitro maturation of mammalian oocytes.
2. Description of Related Art
a. Ovary
The mammalian ovary is responsible for the production of mature oocytes from germ cells and the production of hormones that permit the development of secondary sexual characteristics and the successful completion of pregnancy. The ovary is roughly divided into an outer cortex and an inner, vascular medulla. The stroma of the ovary spans both the cortex, which contains the ovarian follicles in various stages of development, and medulla regions.
A mature follicle is a highly complex unit having certain distinct cell types and consists of several layers of somatic cells surrounding a fluid-filled cavity “antrum” in which resides a single oocyte bathed by follicular fluid. The follicle provides the nutrients and regulatory signals required for oocyte growth and maturation.
Oocytes present in the adult ovary develop from a definite number of primordial germ cells (PGC) that migrate from extragonadal sites to the gonadal ridge to form the primitive ovary during fetal development. Once established in the developing ovary, the proliferating PGC begin to differentiate into oogonia, which are the stem cells that give rise to all the oocytes in the ovary. The population of oogonia goes through a predetermined, species-specific number of mitotic cycles until the cells enter the prophase of meiosis and become oocytes. Meiosis becomes arrested at the diplotene stage of prophase and remains at that stage until folliculogenesis begins at puberty. The meiosis-arrested primary oocytes are contained within a primordial follicle.
b. Primordial Follicles
Primordial follicles are the fundamental developmental units of the mammalian ovary. The number of primordial follicles is determined during early life and most of them remain in a resting state. The store of primordial follicles is not renewable and serves the entire reproductive life span of the adult. Before and throughout the reproductive life of the female, a number of these primordial follicles leave the resting state and start to grow (initial recruitment). The follicles develop to the antral stage where most undergo atresia; however, some of these follicles are rescued (cyclic recruitment) to reach the preovulatory stage. The end of normal reproductive life tyically occurs when the pool of resting primordial follicles is exhausted.
c. Folliculogenesis
Folliculogenesis is the process responsible for the development of ovulatory follicles and the release of one or more mature oocytes at a fixed interval throughout the reproductive life of a female. Folliculogenesis is resumed after a long quiescent phase and involves sequential subcellular and molecular transformations by various components of the follicle. During postnatal life, ovarian follicles continue to grow, mature and either ovulate or regress. Follicles are recruited continuously until the original store is exhausted.
Primordial follicles are activated to become primary follicles. Although oocytes from primordial and primary follicles are not significantly different in size, important changes take place during the primary follicle stage. The corona radiata develops gap junctions with the oocyte and the zona pellucida begins to form between the two cell types. The zona pellucida will not completely surround the oocyte until the follicle reaches the late preantral stage. Secondary follicles begin to appear when the follicular cells of the primary follicles undergo intensive mitotic division. A secondary follicle contains at least two layers of granulosa cells with the theca cells identifiable outside the basement membrane and the follicle contains a fine capillary network. Tertiary or antral follicles are characterized by the presence of a cavity known as antrum, which is filled with follicular fluid. The first antral follicles have an extensive network of gap junctions that permits the transfer of nutrients and regulatory signals between the oocyte and the granulosa cells. Antral follicles develop until they reach the preovulatory size. Inside the antral follicle, cumulus cells surround the oocyte. In vivo, expansion of the cumulus-oocyte complex (COC) is induced after the LH surge at the endpoint of ovulation in preparation for fertilization of the oocyte.
d. Oocyte Maturation
Oocyte maturation is a complex phenomenon during which the oocyte progresses from the diplotene to the metaphase II stage (nuclear maturation) in response to the ovulatory LH surge. Once reaching the metaphase II stage, the oocyte remains arrested until fertilization takes place and the oocyte completes meiosis and forms the pronucleus. Oocyte maturation also involves transformations at the cytoplasmic level that prepare the cell to support fertilization and early embryonic development (cytoplasmic maturation). The final steps of oocyte maturation are crucial to the acquisition of functional properties necessary for further development.
e. In Vitro Fertilization
In vitro fertilization (IVF) of human oocytes is a widely practiced medical technique used to overcome various forms of female and male infertility thereby opening a vast new frontier of research and treatment for the infertile couples. Despite the success of IVF, there is a significant need for improved methods of infertility treatment because about one out of five couples are unable to achieve a successful pregnancy using current IVF treatments.
When IVF was first performed, one mature unfertilized oocyte was removed from the ovary just prior to ovulation. The mature oocyte was fertilized in a laboratory dish (in vitro) and the resulting embryo was transferred back to the woman's uterus. However, it was found that if more oocytes were available for fertilization, there were more embryos available for transfer to the uterus and this significantly increased the pregnancy rate. Therefore, the current clinical practice involves giving patients hormone injections in order to induce the maturation of approximately twenty oocytes.
The standard IVF treatment includes a long phase of hormone stimulation of the female patient, e.g. 30 days, which is initiated by administering a gonadotropin releasing hormone (GnRH) agonist or antagonist to suppress the patient's own follicle stimulating hormone (FSH) and luteinizing hormone (LH). This is followed by injections of exogenous gonadotropins, e.g. FSH and/or LH, in order to ensure development of multiple preovulatory follicles. Just prior to ovulation, multiple in vivo-matured oocytes are removed from the ovaries. The isolated mature oocytes are subsequently fertilized in vitro and cultured, typically for three to six days, before transferring the developed embryos back into the uterus at the 4-8 cell stage.
Continuous efforts have been made to optimize and simplify IVF procedures in order to improve the current overall pregnancy rate of about 25% to 35%. Due to the low pregnancy rates, it is common to transfer two to five embryos in an attempt to increase pregnancy rates.
The administration of hormone injections to induce the maturation of many oocytes simultaneously is known as controlled ovarian hyperstimulation (COH). The advantage of COH is the availability of many more mature oocytes for fertilization, which increases the chances of pregnancy. However, the woman undergoing COH must be closely monitored by daily ultrasound examinations of the ovaries and blood hormone measurements because excessive ovarian stimulation may cause ovarian hyperstimulation syndrome (OHSS), which is a serious and potentially fatal condition. COH is not effective for a number of females, including some with polycistic ovary disease.
f. In Vitro Maturation of Oocytes
The side effects associated with COH could be avoided if immature oocytes could be removed from the oocytes and matured in vitro. Mammalian oocytes undergo spontaneous maturation upon removal from the follicle. Although oocytes matured in vitro have rates of nuclear maturation, fertilization and cleavage similar to in vivo matured oocytes, in vitro matured oocytes have significantly lower blastocyst rates and developmental potential.
Early in each menstrual cycle, several oocytes begin to grow in preparation for undergoing maturation and becoming developmentally competent, i.e., competent to be fertilized and develop into a healthy fetus. By approximately the fifth to seventh day of the cycle, one oocyte becomes dominant and continues to grow while the other oocytes are induced to degenerate. Once an oocyte becomes dominant, it grows and undergoes metabolic changes for approximately one week prior to becoming mature at the time of ovulation. Oocytes that do not undergo this growth phase will mature in vitro and can be fertilized, but are less likely to be developmentally competent. Therefore, the optimal time to obtain the largest number of immature oocytes is early in the cycle before any oocytes have begun to degenerate. However, oocytes removed early in the menstrual cycle and matured in vitro, are less likely to be developmentally competent.
Numerous events within the antral follicle affect oocyte maturation and the acquisition of developmental competency, including: (i) interactions between somatic cells of the follicle (in particular cumulus cells) and the oocyte; (ii) the composition of follicular fluid; and (iii) the temperature and vascularity of the follicular environment. Many of these factors change with follicle size and oocyte growth. In contrast, culture conditions for IVM are based on somatic cells that often do not reflect the follicular environment, and/or have complex compositions or additives such as macromolecule supplements that are undefined in nature. Metabolites typically included in IVM media such as glucose, pyruvate, oxygen and amino acids have been shown to have differential influences on oocyte maturation and competency. Manipulation of these factors and application of gained knowledge of the in vivo environment may result in improved in vitro oocyte maturation and overall in vitro embryo production.
g. IL-6-Type Cytokines
The IL (interleukin)-6-type cytokines, which include IL-6, IL-11, LIF (leukemia inhibitory factor), OSM (oncostatin M), CNTF (ciliary neurotrophic factor), CT-1 (cardiotrophin-1) and CLC (cardiotrophin-like cytokine), activate target genes involved in differentiation, survival, apoptosis and proliferation. IL-6-type cytokines bind to plasma membrane receptor complexes containing the common signal transducing receptor chain gp 130 (glycoprotein 130). Signal transduction involves the activation of JAK (Janus kinase) tyrosine kinase family members, leading to the activation of transcription factors of the STAT (signal transducers and activators of transcription) family. Another major signaling pathway for IL-6-type cytokines is the MAPK (mitogen-activated protein kinase) cascade.
Receptors involved in recognition of the IL-6-type cytokines can be subdivided into the non-signalling α-receptors and the signal transducing receptors. The non-signalling α-receptors include, but are not limited to, IL-6Rα, IL-11Rα, and CNTFRα, where R refers to receptor. The signal transducing receptors include, but are not limited to, gp130, LIFR, and OSMR. The signal transducing receptors associate with JAKs and become tyrosine phosphorylated in response to cytokine stimulation. Each of the IL-6-type cytokines is characterized by a certain profile of receptor recruitment that in all cases involves at least one molecule of gp130.
IL-6, IL-11 and CNTF first bind specifically to their respective α-receptor subunits. Here, only the complex of cytokine and α-receptor efficiently recruits the signalling receptor subunits. IL-6 and IL-11 signal via gp130 homodimers. Most other IL-6 type cytokines signal via heterodimers of either gp130 and the LIFR (LIF, CNTF, CT-1 and CLC) or gp130 and the OSMR (OSM). OSM is able to recruit two different receptor complexes: both LIFR-gp130 and OSMR-gp130 heterodimers. LIF and OSM directly engage their signalling receptor subunits without requirement for additional α-receptor subunits.
1) LIF
LIF elicits a diversity of biological effects on many cell types, including embryonic stem cells, primordial germ cells, neurons, adipocytes, hepatocytes, and osteoblasts. LIF affects various endocrine cell types (utero-placenta unit, bone metabolism, adrenal, ovarian, and testicular). The diversity in biological activity is reflected in the various synonyms of LIF, which include hepatocyte stimulating factor III (HSF III; Baumann and Wong, J. Immunol. 143: 1163, 1989); cholinergic nerve differentiation factor (CNDF; Yamamori et al., Science 246: 1412, 1990); melanoma-derived lipoprotein lipase inhibitor (MLPLI; Mori et al., Biochem. Biophys Res. Comm. 160: 1085, 1989); human interleukin for DA cells (HILDA; Moreau et al., Nature 336: 690, 1988); differentiation factor (D-factor; Tomida et al., J. Biol. Chem. 259: 10978, 1984); differentiation inhibitory factor (DIF; Abe et al., J. Biol. Chem. 264: 8941, 1989); differentiation inhibitory activity (DIA; Smith and Hooper, Devel. Biol. 121: 1, 1987); and differentiation retarding factor (DRF; Koopman and Cotton, Exp. Cell. Res. 154: 233, 1984).
LIF plays a central role in the regulation of diverse adult and embryonic systems. In the reproductive systems, LIF is an important cytokine in early pregnancy. Indeed, female LIF knockout mice are infertile because of a defect in the process of embryonic implantation. LIF is present in human follicular fluid and its levels are regulated according to the stage of antral follicle development. LIF levels in follicular fluid are also responsive to human chorionic gonadotropin (hCG). Cultured granulosa cells from mature follicles, but not from immature follicles, exhibit an increase in LIF production after treatment with βhCG (β-human CG), suggesting that LIF might be involved in ovulation and final oocyte development. Furthermore, LIF has been shown to promote the primordial to primary follicle transition in rats.
LIF is also an important factor in the in vitro culturing of embryonic stem (ES) cells and embryonic germ (EG) cells. EG cells or ES cells retain the stem cell phenotype in vitro when cultured on a feeder layer of fibroblasts when cultured in medium conditioned by certain cells or by the exogenous addition of LIF. In the absence of feeder cells, conditioned medium or exogenous LIF, ES or EG cells spontaneously differentiate into a wide variety of cell types.
2) CT-1
CT-1 causes hypertrophy of cardiac myocytes and has pleiotropic effects on various other cell types. Pennica et al. (J Biol. Chem. 1995 May 5; 270(18): 10915-22) disclose that CT-1 inhibited the differentiation of mouse embryonic stem cells. In vitro biological assays indicated that CT-1 was active in assays where LIF was active and vice-versa. These data showed that CT-1 had a wide range of hematopoietic, neuronal, and developmental activities and that it could act via the LIF receptor and the gp130 signalling subunit. Pennica et al. predict that CT-1 should mimic the many in vitro and in vivo effects of LIF.
WO9730146 discloses a method of enhancing the maintenance of pregnancy in a mammal by culturing an embryo in a medium containing CT-1 prior to introduction of the embryo into a mammal. WO9730146 suggests that media containing CT-1 may be suitable for early manipulative procedures on the oocyte/embryo such as in vitro fertilization, embryo splitting and nuclear transfer where survival rates of embryos are low.
3) OSM
Oncostatin M (OSM) is a pleiotropic cytokine produced late in the activation cycle of T-cells and macrophages that has been extensively characterized with numerous activities attributed to it. OSM was originally isolated from conditioned media of a phorbol ester-treated histiocytic lymphoma cell line, U937, based on the ability to inhibit the growth or development of a human melanoma cell line.
OSM binds to three cell surface receptors. OSM binds to a gp130 polypeptide, also known as the IL-6 signal transduction subunit, with a low affinity. In a second, intermediate affinity interaction, OSM and LIF compete for binding to a receptor composed of the low-affinity LIF receptor and gp130. This intermediate affinity receptor complex is capable of signalling and exerting biological effects in vitro. Although this receptor complex is shared by the two cytokines, the affinity of interaction and biological signals delivered by each of the cytokines are distinct. The third receptor recognized by OSM is a high affinity receptor that is not known to bind to other cytokines. The high affinity OSM receptor is composed of gp130 and an affinity-converting subunit that is required for high affinity and functional ligand-receptor binding.
4) IL-6
Interleukin-6 (IL-6) is a multifunctional cytokine that is produced by a variety of cells such as B-cells, T-cells, monocytes, fibroblasts and endothelial cells. IL-6 exhibits several activities relating to the proliferation and/or differentiation of hematopoietic progenitor cells. These activities result from IL-6 acting alone or in combination with other cytokines such as IL-3 and IL-4. Some specific biological effects of IL-6 include terminal differentiation of B-cells, proliferation and differentiation of T-cells, regulation of the acute phase response, growth regulation of epithelial cells, the differentiation of megakaryocytes, and thrombopoiesis. In accordance with these activities and effects, the target cells for IL-6 include B-cells, T-cells, myeloma cells, megakaryocytes, monocytes, early stem cells and hepatocytes.
Though IL-6 is a multifunctional cytokine, the various biological effects it exerts are believed to be initiated by the stepwise interaction of IL-6 with two distinct receptor subunits on a cell. IL-6 first forms a complex with an 80 kD receptor subunit. This complex binds to a non-ligand subunit, which is a membrane glycoprotein designated gp130. The binding of the IL-6-80 kD receptor complex to gp130 results in signal transduction.
5) sIL-6Rα
The receptor system for IL-6 comprises two functionally different chains: a ligand-binding chain (IL-6R) and a non-ligand-binding but signal-transducing chain (gp130). The gp130 chain associates with the IL-6R/IL-6 complex, resulting in the formation of high-affinity IL-6 binding sites and signal transduction. An extracellular, soluble form of the interleukin-6 receptor (sIL-6R) has been shown to mediate the IL-6 signal through membrane-anchored gp130.
6) IL-6/sIL-6Rα Chimera
A complex of sIL-6R and IL-6 (IL-6/sIL-6Rα chimera) can associate with gp130 expressed on both IL-6R-negative and IL-6R-positive cells. This association induces the homodimerization of gp 130 and the activation of the JAK-STAT pathway thereby leading to cellular response.